Slightly modified expression of the gene encoding methyl-CpG binding protein-2 influences impulsivity in mouse and man

5.1 Overview of project IV

The gene encoding methyl-CpG binding protein-2 (MECP2) maps to chromosome Xq28 and is one of the most dosage-sensitive genes transcriptionally regulating genes associated with neuronal functional integrity (Chahrour et al. 2008). Loss-of-function mutations of MECP2 lead to Rett syndrome, characterized by a gender- and mutation-type-dependent array of symptoms ranging from loss of speech and acquired motor skills to autistic symptoms, severe mental retardation and neonatal encephalopathy (Bienvenu et al. 2006). Interestingly, gene duplication can cause very similar symptoms (Ramocki et al. 2010), which points to the necessity of a tight regulation of gene expression. Accumulating evidence from studies with transgenic mouse models of complete loss-of-function, reduced expression or overexpression of Mecp2, further substantiates that certain levels of the protein are required for the neuronal system to maintain its inhibitory and excitatory balance (Ramocki et al. 2008). Very mild overexpression of Mecp2 (~1.5-fold) already disrupts neuronal homeostasis resulting in higher seizure propensity together with alterations in neuronal branching sites and increased spine density (Bodda et al. 2013). Behaviorally, disturbed neuronal homeostasis across different Mecp2 mouse models expresses as abnormal social behavior and increased aggression (Shahbazian et al. 2002, Moretti et al. 2005, Fyffe et al. 2008, Kerr et al. 2008, Samaco et al. 2009, Chao et al. 2010, Pearson et al. 2012, Samaco et al. 2012, Bodda C 2013).

Interestingly, behavioral effects of altered Mecp2 levels vary dependent of the genetic background (C57Bl6 versus FVB) of the mice.

Our first aim of the present study was to explore the impact of the genetic background on basic behaviors, seizure propensity and spontaneous home cage behavior in transgenic mice with mild overexpression of Mecp2. Indeed, increased aggression in transgenic mice as compared to wildtype littermates could only be detected for the FVB/N background but not for C57Bl6/N mice. We further observed that while mild Mecp2 overexpression in FVB and C57Bl/6 mice left most basic behaviors unaltered, it modulated seizure propensity (gender-dependent) and home cage behavior independent of the genetic background.

In humans, data on aggression/impulsivity in Rett or MECP2 gene duplication syndrome are scarce. A family study characterizing the neuropsychiatric phenotypes of 9 males and 9 females carrying MECP2 duplications revealed a high prevalence of hostility (63%) in the carrier females. Strikingly, females exhibited mild psychiatric symptoms despite 100%

skewing of inactivation of the mutation carrying allele and normal MECP2 mRNA levels (Ramocki et al. 2009). Moreover, one of three very mild cases of Rett syndrome who carried a mutation located in the deletion hotspot of the 3’ end of the MECP2 gene, has been reported to experience episodes of uncontrolled aggression (Huppke et al. 2006). Consequently, also in humans, very mild loss and gain of function of MECP2 might result in a range of neuropsychiatric symptoms including aggression.

Surprisingly, despite the obvious importance of MECP2 for nervous system development and function, the effect of common genetic variations of this gene on human behavior remains widely unknown. It was previously shown that common genetic variants such as 3’ UTR SNP rs2734647 of MECP2 alter the risk for autism (Loat et al. 2008). Moreover, carriers of the minor allele of SNP rs2239464 have decreased cortical surface area in brain regions such as the cuneus (Joyner et al. 2009). Interestingly, this brain region has been shown to be associated with inhibitory control in patients suffering from bipolar disorder (Haldane et al.

2008). In the framework of the present project, we wondered whether slight MECP2 expression differences mediated by common genetic variations would also be associated with aggression relevant behavioral phenotypes in humans and how possible genotype-phenotype relationships would be mechanistically mediated.

Two MECP2 SNPs, rs2239464 and rs2734647, were considered for the phenotype-based association study (inclusion criterion r²<0.8). Translating our findings from the Mecp2 overexpressing mouse model to humans, we selected impulse control, excitement and uncooperativeness (Positive and Negative Syndrome Scale, PANSS) as aggression equivalents from the GRAS data collection (Ribbe et al. 2010). Our PGAS approach was applied to the three target measures and possible sociodemographic and clinical confounders such as age and educational level. Significant genotype-dependent group differences for both selected SNPs were shown for male patients with respect to poor impulse control while only nominal significances/tendencies could be observed for excitement and uncooperativeness. In women, no statistically significant results were obtained. Importantly, no genotype differences withstanding correction for multiple testing were observed for the

sociodemographic and clinical measures. In terms of functional implications of the 3’UTR SNP rs2734647, we identified two microRNAs (miR) with seed binding sites including the 3’UTR SNP position. In vitro luciferase assays demonstrated a loss of down-regulation upon co-transfection with these miRs in case of the allele associated with higher impulsivity. A co-evolutionary effect on one specific miR sequence, adapting for binding to the respective 3'UTR sequence in mouse and man, may emphasize the importance of this regulatory interaction. As a conclusion, well in line with the aggression phenotype observed in transgenic FVB mice, already mildly elevated MECP2 levels seem to be sufficient to modulate impulsive/aggressive behavior in human subjects.

5.2 Manuscript in preparation

Tantra, M.*, Hammer, C.*, Kästner, A.*, Begemann, M., Bodda, C., Stepniak, B., Castillo Venzor, A., Erbaba, B., Tarami, A., Hammerschmidt, K., Schulz-Schaeffer, W., Mannan, A.

and Ehrenreich, H. (In preparation). "Slightly modified expression of the gene encoding methyl-CpG binding protein-2 influences behavior of mouse and man."

*These authors contributed equally to the work.

Personal contribution

I was responsible for the operationalization of impulsivity and aggression in the human sample. I developed the severity scoring of aggression based on the patients’ medical reports and a clinical interview contained in the examination booklet of the GRAS study. I supervised the student performing the aggression scoring and database entering for all schizophrenic patients. Moreover, I performed the human PGAS data analyses and assisted in the interpretation of the results. I designed the tables and participated in the conception, design, drafting, revision and publication of the manuscript.

6. Operationalization and validation of an autistic phenotype in the GRAS